Transcription is the synthesis of a strand of RNA representing or complementary to the coding strand of a DNA duplex. It takes place by the usual process of complementary base pairing and is catalyzed by the enzyme RNA polymerase. Transcription is the first step in the expression of a gene and it is the principle step at which gene expression is controlled or regulated.
The transcription process can be controlled at any one of the three stages of transcription: initiation, elongation, or termination. In eukaryotic systems, initiation involves the association of the RNA polymerase with several other enzymes and factors at the promoter. For accurate initiation, a number of the specific initiation factors are required.
Elongation is the phase in which the RNA polymerase moves along the strand of DNA, extending the growing RNA chain as it does so. As the RNA polymerase moves it unwinds the DNA helix to expose a new segment of the template in single-stranded form. Nucleotides are then covalently added to the 3' end of the elongating RNA chain forming an RNA-DNA hybrid in the unwound region. The DNA strand then re-associates with its DNA complement, thereby reforming the double helix structure and displacing the single-stranded RNA strand. Thus, elongation involves the transient disruption of DNA structure to form an unwound region that exists as a hybrid RNA-DNA duplex and a displaced single strand of DNA.
Termination of transcription occurs when the RNA polymerase reaches a termination codon, i.e., a noncoding segment of the DNA template strand. At this point, no additional nucleotides are added to the RNA chain. The termination stage ends when the RNA polymerase, DNA template, and newly synthesized RNA chain dissociate into separate entities.
Messenger RNA (mRNA)transcription is a complex biochemical process requiring the action of multiple transcription factors. Transcription factors include both initiation and elongation factors, which control the activity of the RNA polymerase at the initiation and elongation stages of transcription, respectively. Several of these factors are known to be essential for initiation and are referred to as factors D, E, A, G, and B from Saccharomyces cerevisiae, .tau., .alpha., .beta..gamma., .delta., and .epsilon. from rat liver, and TFIID, TFIIB, RAP30/74 or TFIIF, BTF2 or TFIIH, and TFIIE from human cells.
In addition to these factors, other proteins have been shown to stimulate either the initiation or elongation stages of transcription by RNA Polymerase II. One such factor, designated TFIIA, has been purified from both Saccharomyces cerevisiae and mammalian cells. TFIIA appears to promote assembly of the transcriptional pre-initiation complex. Although TFIIA is not essential for initiation, several lines of evidence suggest that it functions to increase the number of pre-initiation complexes that form at the promoter.
Considerable progress has recently been achieved identifying and characterizing transcriptional factors that support a basal level of transcription by RNA Polymerase II. Significantly less information, however, is available on transcription factors regulating the efficiency of transcriptional initiation or RNA chain elongation. Such transcriptional factors play an important role in regulating gene expression.
Currently, five general transcription elongation factors influencing RNA chain elongation have been identified and characterized with a high degree of certainty. These are SII, P-TEFb, TFIIF, ELL, and Elongin (also known as "SIII"). The general elongation factors TFIIF (RAP30/74) and ELL act to increase the overall rate of RNA chain elongation by suppressing transient pausing of the RNA polymerase at a variety of sites. The transcription factors SII and P-TEFb prevent RNA Polymerase II from arresting transcription prematurely. SII has been shown to promote RNA polymerase read-through at intrinsic pause sites in a human histone gene, the adenyl virus genome, and at several other sites. SII is a 38 kiloDalton (kD) elongation factor that promotes passage of RNA Polymerase II through transcriptional impediments such as nucleoprotein complexes and DNA sequences acting as intrinsic arrest sites. P-TEFb catalyzes the conversion of early, termination prone elongation complexes into productive elongation complexes.
A fifth elongation factor which increases the overall rate at which RNA Polymerase II transcribes DNA is Elongin. Elongin is a trimeric complex consisting of three protein subunits labeled Elongin A (110 kD as measured by SDS-PAGE), Elongin B (18 kD as measured by SDS-PAGE), and Elongin C (15 kD as measured by SDS-PAGE) having 773, 118 and 112 amino acid residues, respectively. Elongin A is capable of weakly stimulating transcriptional activity at a low level in the absence of Elongin B and/or C, while Elongins B and C serve regulatory functions which increase the transcriptional activation activity of Elongin A. Elongins B and C bind stably to each other in the absence of Elongin A to form a binary complex (Elongin BC) that interacts with Elongin A strongly inducing its transcriptional activity.
In addition, it has been shown that Elongin C can assemble with Elongin A in the absence of Elongin B to form an Elongin AC complex which increased specific activity, thereby increasing the rate of RNA chain elongation. Elongin B does not interact with Elongin A in the absence of Elongin C and apparently functions like a chaperone protein facilitating the assembly and enhancing the stability of the Elongin ABC complex. The identification, purification and characterization of Elongin and its subunits Elongins A, B and C have been described in U.S. Ser. No. 08/524,757 filed on Sep. 7, 1995, now U.S. Pat. No. 5792 634 and incorporated herein by reference.
Elongin has also been reported to interact with the product ("pVHL") of the Von Hippel-Lindau tumor suppressor gene. The Von Hippel-Lindau tumor suppressor gene, which predisposes individuals to various tumor types, translates into a 213 amino acid protein capable of binding to and inhibiting the activity of Elongin. In particular, it has been reported that wild-type pVHL binds tightly and specifically to the Elongin BC complex and prevents it from activating Elongin A. That is, binding of the PVHL protein and Elongin A to the Elongin BC complex are mutually exclusive in vitro.
The previous work on Elongin described above provided a useful product for regulating the transcriptional activity of RNA Polymerase II. There still remained, however, a need to identify the functional domains of the Elongin subunits. As described herein, the Elongin A transcriptional activation domain has now been identified. This domain has been found to define a new evolutionarily conserved class of inducible activation domain. It has also been shown that the transcriptional activation domain of Elongin A and the Von Hippel-Lindau tumor suppressor protein (pVHL) interact with the Elongin BC complex through a conserved Elongin BC binding site motif essential for induction of Elongin A activity by Elongin BC and for tumor suppression by the VHL protein. In addition, the regions of Elongin C important for binding to Elongin B and for binding to and activating Elongin A have also been identified.
The identification and characterization of these domains serves two purposes. First, Elongin subunits or fragments thereof having at least the functional domains described herein can be used in place of the entire subunits in in vitro transcriptional assays or systems. Secondly, and perhaps more importantly, these regions can be used as laboratory reagents for work concerning the transcriptional activation activities of Elongin.